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Systematic imaging reveals features and changing localization of mRNAs in Drosophila development.

Jambor H, Surendranath V, Kalinka AT, Mejstrik P, Saalfeld S, Tomancak P - Elife (2015)

Bottom Line: We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary and detectable in all of its cell types-the somatic epithelial, the nurse cells, and the oocyte.Genes defined by a common RNA localization share distinct gene features and differ in expression level, 3'UTR length and sequence conservation from unlocalized mRNAs.Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

ABSTRACT
mRNA localization is critical for eukaryotic cells and affects numerous transcripts, yet how cells regulate distribution of many mRNAs to their subcellular destinations is still unknown. We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary and detectable in all of its cell types-the somatic epithelial, the nurse cells, and the oocyte. Genes defined by a common RNA localization share distinct gene features and differ in expression level, 3'UTR length and sequence conservation from unlocalized mRNAs. Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types. This genome scale image-based resource (Dresden Ovary Table, DOT, http://tomancak-srv1.mpi-cbg.de/DOT/main.html) enables the transition from mechanistic dissection of singular mRNA localization events towards global understanding of how mRNAs transcribed in the nucleus distribute in cells.

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FISH screen results and controls.(A) Estimate of false-positive/negative rate of the in situ screen using comparison with the independent transcriptomics data. A gene was classified as falsely positive if it was annotated as ubiquitous or specific by FISH but was not detectable by either 3Pseq or RNAseq at any time-point of oogenesis. In 20% of the experiments we failed to detect in situ signal (‘no signal’) although the transcript was detected at least at one time point by at least one deep sequencing method. These may represent false negative results, possibly due to non-functional RNA probes, however, we nevertheless included them in the downstream analysis in the no signal category. (B) mRNA enrichments in the somatic epithelial cells overlaying the oocyte (CG14639) and at the cortex of nurse cells (Actn). RNA signal shown in green. DNA, labelled with DAPI, is shown in magenta. Scale bar 30 μm. (C) CG9609 and Doa mRNAs detected in the oocyte nucleus showing the enrichment over time at stages 9, 10A and 10B. At stage 9 only few small mRNA foci are visible, at stage 10 the mRNAs were enriched in proximity of the DNA in two large foci (see arrows). (D) Karyogram showing the chromosomal position of genes for nuclear, anterior, and posterior RNA localization classes. Neither nuclear RNA genes, which often appear in foci-like enrichments nor anterior or posterior class genes appear clustered on the chromosome. (E) Examples of FISH experiment detecting distributions of non-coding RNA (in green). While pri-miRNA-318 is enriched in somatic epithelial cell nuclei, pri-miRNA-303, pri-miRNA-31-b and the long non-coding RNA CR42862 are restricted to nuclei of the germline nurse cells. Scale bar 30 μm, DNA in magenta.DOI:http://dx.doi.org/10.7554/eLife.05003.007
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fig2s1: FISH screen results and controls.(A) Estimate of false-positive/negative rate of the in situ screen using comparison with the independent transcriptomics data. A gene was classified as falsely positive if it was annotated as ubiquitous or specific by FISH but was not detectable by either 3Pseq or RNAseq at any time-point of oogenesis. In 20% of the experiments we failed to detect in situ signal (‘no signal’) although the transcript was detected at least at one time point by at least one deep sequencing method. These may represent false negative results, possibly due to non-functional RNA probes, however, we nevertheless included them in the downstream analysis in the no signal category. (B) mRNA enrichments in the somatic epithelial cells overlaying the oocyte (CG14639) and at the cortex of nurse cells (Actn). RNA signal shown in green. DNA, labelled with DAPI, is shown in magenta. Scale bar 30 μm. (C) CG9609 and Doa mRNAs detected in the oocyte nucleus showing the enrichment over time at stages 9, 10A and 10B. At stage 9 only few small mRNA foci are visible, at stage 10 the mRNAs were enriched in proximity of the DNA in two large foci (see arrows). (D) Karyogram showing the chromosomal position of genes for nuclear, anterior, and posterior RNA localization classes. Neither nuclear RNA genes, which often appear in foci-like enrichments nor anterior or posterior class genes appear clustered on the chromosome. (E) Examples of FISH experiment detecting distributions of non-coding RNA (in green). While pri-miRNA-318 is enriched in somatic epithelial cell nuclei, pri-miRNA-303, pri-miRNA-31-b and the long non-coding RNA CR42862 are restricted to nuclei of the germline nurse cells. Scale bar 30 μm, DNA in magenta.DOI:http://dx.doi.org/10.7554/eLife.05003.007

Mentions: Based on our in situ hybridization screen, we identified 3475 mRNAs as being expressed and most of these mRNAs were also detectable by RNA sequencing. Both sequencing techniques were in good agreement with each other (Figure 1A, Figure 2—figure supplement 1A, Figure 5—figure supplement 1A). Of the expressed genes, 64% showed ubiquitous mRNA distribution in ovary cells throughout oogenesis (ubiquitous), but we also observed mRNA expressions restricted to subsets of cells (cellular) and mRNAs that asymmetrically localized in the cytoplasm (subcellular) or to the nuclei of cells (nuclear).10.7554/eLife.05003.003Figure 1.Summary of the fluorescent in situ hybridization (FISH) screen in ovaries.


Systematic imaging reveals features and changing localization of mRNAs in Drosophila development.

Jambor H, Surendranath V, Kalinka AT, Mejstrik P, Saalfeld S, Tomancak P - Elife (2015)

FISH screen results and controls.(A) Estimate of false-positive/negative rate of the in situ screen using comparison with the independent transcriptomics data. A gene was classified as falsely positive if it was annotated as ubiquitous or specific by FISH but was not detectable by either 3Pseq or RNAseq at any time-point of oogenesis. In 20% of the experiments we failed to detect in situ signal (‘no signal’) although the transcript was detected at least at one time point by at least one deep sequencing method. These may represent false negative results, possibly due to non-functional RNA probes, however, we nevertheless included them in the downstream analysis in the no signal category. (B) mRNA enrichments in the somatic epithelial cells overlaying the oocyte (CG14639) and at the cortex of nurse cells (Actn). RNA signal shown in green. DNA, labelled with DAPI, is shown in magenta. Scale bar 30 μm. (C) CG9609 and Doa mRNAs detected in the oocyte nucleus showing the enrichment over time at stages 9, 10A and 10B. At stage 9 only few small mRNA foci are visible, at stage 10 the mRNAs were enriched in proximity of the DNA in two large foci (see arrows). (D) Karyogram showing the chromosomal position of genes for nuclear, anterior, and posterior RNA localization classes. Neither nuclear RNA genes, which often appear in foci-like enrichments nor anterior or posterior class genes appear clustered on the chromosome. (E) Examples of FISH experiment detecting distributions of non-coding RNA (in green). While pri-miRNA-318 is enriched in somatic epithelial cell nuclei, pri-miRNA-303, pri-miRNA-31-b and the long non-coding RNA CR42862 are restricted to nuclei of the germline nurse cells. Scale bar 30 μm, DNA in magenta.DOI:http://dx.doi.org/10.7554/eLife.05003.007
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fig2s1: FISH screen results and controls.(A) Estimate of false-positive/negative rate of the in situ screen using comparison with the independent transcriptomics data. A gene was classified as falsely positive if it was annotated as ubiquitous or specific by FISH but was not detectable by either 3Pseq or RNAseq at any time-point of oogenesis. In 20% of the experiments we failed to detect in situ signal (‘no signal’) although the transcript was detected at least at one time point by at least one deep sequencing method. These may represent false negative results, possibly due to non-functional RNA probes, however, we nevertheless included them in the downstream analysis in the no signal category. (B) mRNA enrichments in the somatic epithelial cells overlaying the oocyte (CG14639) and at the cortex of nurse cells (Actn). RNA signal shown in green. DNA, labelled with DAPI, is shown in magenta. Scale bar 30 μm. (C) CG9609 and Doa mRNAs detected in the oocyte nucleus showing the enrichment over time at stages 9, 10A and 10B. At stage 9 only few small mRNA foci are visible, at stage 10 the mRNAs were enriched in proximity of the DNA in two large foci (see arrows). (D) Karyogram showing the chromosomal position of genes for nuclear, anterior, and posterior RNA localization classes. Neither nuclear RNA genes, which often appear in foci-like enrichments nor anterior or posterior class genes appear clustered on the chromosome. (E) Examples of FISH experiment detecting distributions of non-coding RNA (in green). While pri-miRNA-318 is enriched in somatic epithelial cell nuclei, pri-miRNA-303, pri-miRNA-31-b and the long non-coding RNA CR42862 are restricted to nuclei of the germline nurse cells. Scale bar 30 μm, DNA in magenta.DOI:http://dx.doi.org/10.7554/eLife.05003.007
Mentions: Based on our in situ hybridization screen, we identified 3475 mRNAs as being expressed and most of these mRNAs were also detectable by RNA sequencing. Both sequencing techniques were in good agreement with each other (Figure 1A, Figure 2—figure supplement 1A, Figure 5—figure supplement 1A). Of the expressed genes, 64% showed ubiquitous mRNA distribution in ovary cells throughout oogenesis (ubiquitous), but we also observed mRNA expressions restricted to subsets of cells (cellular) and mRNAs that asymmetrically localized in the cytoplasm (subcellular) or to the nuclei of cells (nuclear).10.7554/eLife.05003.003Figure 1.Summary of the fluorescent in situ hybridization (FISH) screen in ovaries.

Bottom Line: We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary and detectable in all of its cell types-the somatic epithelial, the nurse cells, and the oocyte.Genes defined by a common RNA localization share distinct gene features and differ in expression level, 3'UTR length and sequence conservation from unlocalized mRNAs.Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

ABSTRACT
mRNA localization is critical for eukaryotic cells and affects numerous transcripts, yet how cells regulate distribution of many mRNAs to their subcellular destinations is still unknown. We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary and detectable in all of its cell types-the somatic epithelial, the nurse cells, and the oocyte. Genes defined by a common RNA localization share distinct gene features and differ in expression level, 3'UTR length and sequence conservation from unlocalized mRNAs. Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types. This genome scale image-based resource (Dresden Ovary Table, DOT, http://tomancak-srv1.mpi-cbg.de/DOT/main.html) enables the transition from mechanistic dissection of singular mRNA localization events towards global understanding of how mRNAs transcribed in the nucleus distribute in cells.

Show MeSH
Related in: MedlinePlus